towards improved death receptor targeted therapy for ... - TI Pharma

MATERIALS & METHODS
TRAIL‐induced migration and invasion
Cell lines and chemicals
NSCLC cells, H460, H322, SW1573, and A549, derived from ATCC in 2003, were cultured
as monolayers in RPMI 1640 medium, supplemented with 10% (v/v) FBS, 100 units/ml
penicillin, and 100 µg/ml streptomycin. Cells were maintained in a humidified 5% CO2
atmosphere at 37°C. The cell lines were tested for their authenticity by short tandem
repeats (STR) profiling DNA fingerprinting (Baseclear, Leiden, The Netherlands). PP2,
PD098059 (Sigma‐Aldrich, St. Louis, MO, USA), dasatinib and saracatinib (both from LC
Laboratories, Woburn, MA, USA) were dissolved in DMSO to 20 mM stock solutions.
LY294002 (Sigma‐Aldrich) was dissolved in DMSO to 10 mM stock solution.
MTT assay
A total of 10,000 cells were seeded in 96‐well plates (Greiner Bio‐One, Frickenhausen,
Germany). The next day 100 µl medium with or without TRAIL was added with increasing
concentrations to the cells. After 24 h incubation, the medium was discarded and 50 µl of
a MTT solution (0.5 mg/ml (Sigma‐Aldrich) in HBSS) was added and incubated at 37°C for
1.5 h. The formazan crystals were dissolved using 150 µl dimethyl sulfoxide (DMSO) and
absorbance was measured at 540 nm (Tecan, Männedorf, Switzerland). Results are
presented as percentage of viable cells taking the control (untreated cells) as 100%
survival. The concentration resulting in 50% of cell growth inhibition (IC50) was derived
from the growth inhibition curve.
Receptor cell surface expression
Analysis of TRAIL‐receptor membrane expression was performed using a flow cytometer
(Epics Elite, Coulter‐Electronics, Hialeah, FL, USA). Adherent cells were harvested by
treatment with trypsin and washed twice in PBS containing 1% BSA. Appropriate
concentrations of antibodies dissolved in PBS/1% BSA were added to the cells. The
following antibodies were used to determine TRAIL receptor membrane expression:
TRAIL‐R1 (HS101), TRAIL‐R2 (HS201), TRAIL‐R3 (HS301), TRAIL‐R4 (HS402), all from Alexis.
Mouse IgG (DAKO) was used as isotype control. Subsequently, cells were incubated for
30 min on ice, washed twice with cold PBS/1% BSA, and incubated with FITC‐conjugated
rabbit‐antimouse (DAKO, Glostrup, Denmark) for 30 min on ice. After washing, the cells
were analyzed by flow cytometry. Surface expression is shown as a ratio of the signal of
the specific TRAIL‐receptor antibody and the negative isotype control antibody.
Migration assays
Cell migration was determined using the wound healing assay as described previously
[13]. In brief, NSCLC cells were seeded in 96‐wells plates and grown till confluence. A 96
well floating‐pin transfer device with a pin diameter of 1.58 mm coming to a flat point at
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